Incorporation of exogenous lactic bacteria into the oral microflora

ABSTRACT

Compositions for the prophylaxis or treatment of dental caries, dental plaque, and periodontal infection that include lactic bacteria that are not part of the resident microflora of the mouth, that are low acidifying, and that are capable of adhering directly to the pellicle of the teeth. The compositions are used in methods of treating or preventing dental caries, dental plaque, and periodontal infection.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of the U.S. national phasedesignation of PCT application no. PCT/EP99/05473, filed Jul. 26, 1999,the entire contents of which are incorporated herein by referencethereto.

FIELD OF THE INVENTION

The present invention relates to the incorporation of exogenous lacticbacteria into the oral microflora for the prophylaxis or the treatmentof dental caries, dental plaque, and periodontal infection.

BACKGROUND OF THE INVENTION

The mouth (oral cavity) contains resident and non-resident microflora.The resident microflora includes microorganisms that are able toestablish a more or less permanent residence on the oral surfaces. Thesebacteria are mainly localized on the tongue, the buccal mucosa, and theteeth while the gingiva, lips, checks, palate, and floor of the mouthonly support a very sparse microflora.

On the tongue and the buccal mucosa, the natural resident microfloraincludes microorganisms selected from Streptococcus, Veillonella,Bacteroides, and Haemophilus. On the teeth, Streptococci, Lactobacilliand Actynomyces predominate but a variety of Gram positive and negativecocci and rods can be also found.

For example, Frandsen et al. showed that S. sanguis predominates on thebuccal mucosa but its primary habitat is the surface of teeth, that S.gordonji grows in the mature supragingival plaque, and that S. oralisand S. mitis grow in the initial dental plaque (Oral Microbiol.Immunol., 6, 129-133, 1991). Strains belonging to the mutans group arelocalized on teeth (S. criscetus, S. downei, S. ferus, S. macacae, S.mutans, S. rattus, S. sobrinus). Strains belonging to the S. millerigroup predominate in dental abscesses (S. anginosus, S. constellatus, S.intermedius) (Bentley et al., Int. J. System. Bacter. 1991, 41, 487-494;Wood et al., The Genera of Lactic Acid Bacteria, Blackie Academic andProfessional, Chapman & Hall, W. H. eds., 1995).

Many of these microorganisms are innocuous commensal microorganisms, buta lot of them have been recognized as being the etiologic agentresponsible for several diseases (Hill, M. J. and Marsh, P. D. eds.Human Microbial Ecology, 1990, CRC Press, Boca Raton Fla., USA).

Dental plaque is a film that forms on the surface of teeth consisting ofbacterial cells in a matrix of extracellular polysaccharide and salivaryproducts. Immediately after eruption, the teeth are covered with anamorphous layer of saliva, the acquired enamel pellicle (AEP), that isabout 1.3 μm thick and cannot be removed by normal tooth brushing. Thedeposition of bacteria on teeth immediately follows the formation of theAEP and plaque becomes evident in 8-12 hours as a multi-layeredstructure. The first layer consists of bacteria (earliest colonizers)that attach to teeth, mainly via specific adhesion-receptor recognition,and forms a substratum for the second colonizers that adhere one to theother by analogous specific binding or by simple juxtaposition. Plaquecohesion is essentially guaranteed by three mechanisms: the presence ofa salivary pellicle on the outer bacteria layer, the specificco-aggregation among the different bacterial species, and the glucanssynthesized by the bacteria that remain entrapped in the plaque matrix(Skopek et al., Oral Microbiol. Immunol., 2, 19-24, 1994; Kolenbranderet al., Meth. Enzymol., 253, 385-397, 1995; Hiroi et al., FEMS MicrobiolLett., 96, 193-198, 1992; Gibbons et al., Infect. Immun., 52, 555-561,1986).

The organic acids produced by oral bacteria during the fermentationprocess directly cause dental caries. These acids attack the hard tissueof teeth with the consequent release of ions such as calcium, phosphate,carbonate, magnesium, fluoride, and sodium. When the pH in the oralcavity again increases to around neutrality, the saliva becomessaturated with calcium so that calcium liberation from the tooth isprevented. Among all the food residues found in the mouth, carbohydratesshow the highest caries promoting effect since they are directlyavailable for fermentation by oral bacteria.

Potentially all microorganisms that ferment sugars are cariogenic, butthe primary etiological agents of coronal and root caries are the mutansstreptococci because they are strong acid producers; Lactobacilli, thatare highly aciduric, however, can also be implicated. In humans, S.mutans and S. sobrinus are the more cariogenic strains, and live onteeth while not colonizing the entire dentition. Their number is alsoless on anterior teeth than on molar teeth (Lindquist et al., Dent.Res., 69, 1160-1166, 1990). Moreover in human approximal plaque, S.mutans and S. sobrinus preferentially colonize the most caries-pronesite apical to the contact area (Ahmady et al., Caries Res., 27,135-139, 1993). A higher prevalence of S. sobrinus was also found in themolar regions compared with that of S. mutans (Lindquist et al., CariesRes., 25, 146-152, 1991).

S. mutans and S. sobrinus have been shown to attach to the pellicle ofteeth mainly via specific adhesion-receptor interaction. Gibbons et al.showed that S. mutans carries an adhesion which binds to salivarycomponents in the pellicle, while S. sobrinus cells appear to possess anadhesion which binds to glucan in the pellicle (Infect. Immun., 52,555-561, 1986).

The transient microflora comprise exogenous bacteria that areoccasionally present in the mouth, but that do not establish a permanentresidence therein (even if repeated oral administrations of thesebacteria are carried out). All the food bacteria, and in particularlactic acid bacteria, can be part of this transient microflora. Theseexogenous lactic bacteria have never been shown to be capable ofdirectly adhering to the pellicle of teeth. Repeated administration ofexogenous lactic bacteria may, however, lead to colonization of themouth on all the oral surfaces, such as the tongue, the buccal mucosa,the gingiva, lips, cheeks, palate, floor, and the teeth. Thiscolonization may result from attachments via specific bindings tobacteria of the resident microflora (co-aggregation phenomena), viaentrapment in the matrix of polysaccharide produced by the residentbacteria, or via adhesion to saliva proteins (especially glycoproteins).

Lactobacillus casei rhamnosus GG (ATCC53103) has been reported tocolonize the mouth, most probably on the epithelium of the buccalmucosa. This strain also adheres to the epithelium of the intestinaltract (U.S. Pat. No. 5,032,399, Gorbach et al.; Micr. Ecol. In Healthand Dis., 2, 295-298, 1994). By contrast L. rhamnosus does not adhere toteeth.

Japanese patent no. 4021633 (Cyconmedix KK) also reported colonizationof the mouth by Lactobacillus acidophilus, most probably on theepithelium of the buccal mucosa. Many Lactobacillus acidophilus areknown to also adhere to the epithelium of the intestinal tract(EP577904; EP199535; Perdigon et al., Medicina, 46, 751-754, 1986;Perdigon et al., Immunology, 63, 17-23, 1988).

Exogenous bacteria can also produce factors that inhibit the growth ofthe resident microflora in the mouth. For example, EP759469 (Société desProduits Nestle) described the use of a bacteriocin produced byMicrococcus varians for inhibiting the development of the oral pathogensS. sobrinus, S. sanguis, S. mutans, and A. viscosus.

There are several strategies to minimize the development of residentmicroflora of the mouth. For example, by administering commensalbacteria of the resident microflora that are not cariogenic, such asStreptococcus salivarius and/or Stomatococcus mucilaginosus, and/orrepeated administration of exogenous lactic bacteria such as L. casei,L. fermentum, L. acidophilus, L. crispatus, L. gasseri, L. salivarius,L. bulgaricus, and S. salivarius (Tanzer et al., Infec. and Immunity,48,44-50, 1985; WO92/14475).

The application of bacteriocins is another investigated strategy whichhas been used to reduce tooth caries. These molecules have attractedinterest as prospective anti-carie agents and as factors important inmodulating colonization of the oral cavity. The anti-carie potential ofapplying bacteriocins comes from their potent and broad antibacterialactivity against mutans streptococci and bacteria associated with dentalplaque and their natural occurrence in bacteria regarded as being safeto humans (U.S. Pat. No. 5,368,845 to Colgate, and WO 94/12150 toSmithkline Beecham).

The application of milk derivatives is also of interest for the healthof the mouth. Indeed, U.S. Pat. No. 5,427,769 (Nest{umlaut over (e )}S.A.) describes another alternative wherein dental caries are prevented bycontacting teeth with an edible composition containing micellar caseinin amount sufficient to inhibit colonization by Streptococcus sobrinus.EP748591 (Societe des Produits Nestle S. A.) also reports the use offluoridated micellar casein or its micellar subunits for treating dentalcaries or plaque. U.S. Pat. No. 4,992,420 (Nestec S. A.) describestreatment of the buccal cavity with kappa-caseino-glycomacropeptidederived from milk for eradicating plaque and caries.

Lactic bacteria that are not part of the resident microflora of themouth have never been shown to be really capable of directly adhering tothe pellicle of teeth. By colonizing the surface of teeth, however, suchlactic bacteria could exert an inhibitory activity against the growth ofthe resident microflora, including oral pathogens.

SUMMARY OF THE INVENTION

The present invention is directed to a method of treating or preventingdental caries, dental plaque, and periodontal infection in a humans oranimals comprising administering to the oral cavity of a human or animalone or more lactic bacteria that are not part of the resident microfloraof the mouth, that are low acidifying, and that are capable of adheringdirectly to the pellicle of the teeth to displace from the teeth orprevent attachment to the teeth of cariogenic strains of bacteria thatare resident microflora of the mouth. In one embodiment the lacticbacteria to be administered provides a pH in the oral cavity of about5.5 to 5.7. Advantageously, the lactic bacteria may be of dairy origin.

The lactic bacteria is preferably one or more of Streptococcusthermophilus, Lactococcus lactis subsp. lactis, or Lactococcus lactissubsp. lactis biovar diacetylactis. In particular the lactic bacteria isone of the strains CNCM I-1984, CNCM I-1985, CNCM I-1986, CNCM I-1987,and LMG P-18997.

Preferably, the lactic bacteria has optimal growth at a temperature ofabout 37° C., i.e., the temperature of the mouth. The lactic bacteriamay have been genetically modified to have improved adherence to thepellicle of the teeth or to be less acidifying than resident microflorafound in the mouth. The lactic bacteria may be genetically modified tohave improved adherence to the pellicle of the teeth by insertion of theX17390 gene, the X14490 gene, or the X53657 gene.

In another embodiment the method of the invention further involvesadministering the lactic bacteria in combination with one or more ofmilk, fermented milk, milk derivatives, or bacteriocin. The milkderivative may be one or more of a caseino-glycomacropeptide, micellarcasein, fluorinated micellar casein, or renneted milk.

The invention also relates to dental compositions for use in the methodsof the invention. The lactic bacteria may be present in thesecompositions in an amount of 10⁴ to 10⁹ cfu/g in order to provide a pHof at least 5.5 when the composition is administered to the mouth of ahuman or animal. When bacteriocin is present in an the composition, itis typically present in an mount of 0.00001 to 50 percent by weight ofthe composition. When the milk derivative is one or more of acaseino-glycomacropeptide, micellar casein, fluorinated micellar caesin,or renneted milk it may be present in an amount of at least about 0.1percent by weight of the composition. The composition may furtherinclude one or more of an oil soluble antioxidant in an amount of about0.005 to 0.5 percent by weight of the composition and an abrasive. Thecomposition may be in the form of a toothpaste, mouth rinse, gum, spray,beverage, candy, infant formula, ice cream, frozen dessert, sweet saladdressing, milk preparation, cheese, quark, yogurt, acidified milk,coffee cream, or whipped cream.

The invention also relates to a method for screening lactic bacteriacapable of adhering to teeth. The method involves the steps of preparingmonoclonal antibodies that recognize specific surface proteins of lacticbacteria strains that are capable of adhering to the teeth and screeninglactic bacteria strains with the monoclonal antibody to identify thestrains of lactic bacteria that adhere to teeth.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 represents the adhesion saturation curves for S. sobrinus OMZ 176(1a), L. lactis NCC2211 (1b), and S. thermophilus NCC1561 (1c);

FIG. 2 represents the effect of CGMP on the adhesion to S-HA beads of S.sobrinus OMZ 176, L. lactis NCC2211, and S. thermophilus NCC1561;

FIG. 3 represents the effect of As-CGMP on the adhesion to S-HA beads ofS. sobrinus OMZ 176, L. lactis NCC2211, and S. thermophilus NCC1561.

DETAILED DESCRIPTION OF THE INVENTION

The object of the present invention is to use lactic bacteria that arenot part of the resident microflora of the mouth, that is lacticbacteria that are low acidifying and that are capable of adheringdirectly to the pellicle of the teeth, to prepare a composition intendedfor the prophylaxis or the treatment of dental caries, dental plaque,and periodontal infection.

In one embodiment of the invention the lactic bacteria have beengenetically modified to increase its adherence to the pellicle of theteeth via adhesion factors and/or genetically modified to be even lessacidifying, contributing to a pH in the oral cavity of about 5.5 to 7.

The lactic bacteria may be selected from the group consisting of:

an acidifying lactic bacteria that adheres to the pellicle of the teethand that has been genetically modified so that it is low acidifyingcompared to resident microflora;

a non adherent lactic bacteria that is low acidifying and that has beengenetically modified so that it adheres to the pellicle of the teeth;

a non-adherent acidifying lactic bacteria that has been geneticallymodified so that it adheres to the pellicle of the teeth and geneticallymodified so that it is low acidifying compared to resident microflora.

In another embodiment the bacteria, that is not part of the residentmicroflora, is low acidifying compared to resident microflora and iscapable of adhering directly to the pellicle of the teeth.

In another embodiment the composition for the health of the mouthcomprises (1) at least a lactic bacteria that is not part of theresident microflora of the mouth, which is capable of adhering directlyto the pellicle of the teeth and contributing to a pH in the oral cavityof above 5.5, and (2) any form of caseinoglycomacropeptide, micellarcasein, fluorinated micellar casein, renneted milk, or bacteriocin.

The invention also provides a method for screening lactic bacteriacapable of adhering to tooth. The method comprises the steps of: (1)preparing monoclonal antibody recognizing specific surface proteins of alactic bacteria strain capable of adhering to the teeth, and (2)screening any lactic bacteria strain by use of the monoclonal antibodyof strain capable of adhering to the teeth.

The term “mouth,” as used herein defines the oral cavity of humans oranimals such as pets, composed by the oral mucosa (gums, lips, cheeks,palate, and floor of the mouth), the tongue, and the teeth (includingartificial structures).

Resident microflora of the mouth includes all microorganisms thatnaturally live in the mouth because they can establish a permanentresidence on the oral surfaces. The resident microflora of the mouthalso includes bacteria that live in the interfacial region between thedental hard and soft tissues (the junction tooth-gingiva), even thoughtthe gingival crevice and the periodontal pocket are not present in ahealthy mouth. This microflora includes microorganisms selected fromStreptococcus, Staphylococcus, Enterococcus, Micrococcus,Peptostreptococcus, Peptococcus, Lactobacillus, Corynebacterium,Actinomyces, Arachnia, Rothia, Alcaligenes, Eubacterium,Propionibacterium, Bifidobacterium, Bacillus, Clostridium,Neisseria/Branhamella, Veillonella, Enterobacteriaceae, Campylobacter,Eikenella, Actinobacillus, Capnocytophga, Haemophilus, Simonsiella,Bacteroides, Fusobacterium, Porphyromonas, Prevotella, Leptotrichia,Wohlinella/Selenomonas, Mycoplasma, Candida, Spirochaetes, Protozoa.

Transient microflora comprises exogenous bacteria that can beoccasionally present in the mouth, but that do not establish a permanentresidence. This transient microflora may comprise all the foodmicro-organisms, such as the bifidobacteria (B. infantis, B.adolescentis, B. breve and B. longum); the lactococci (Lactococcuslactis subsp. lactis, Lactococcus lactis subsp. cremoris, andLactococcus lactis subsp. lactic biovar diacetylactis); the streptococci(Streptococcus thermophilus, S. lactis, S. lactis cremoris and S. lactisdiacetylactis); the Lactobacilli (Lactobacillus delbrueckii subsp.bulgaricus, Lactobacillus helveticus, Lactobacillus farciminis,Lactobacillus alimentarius, Lactobacillus casei subsp. casei,Lactobacillus delbruckii subsp. lactis, Lactobacillus sake,Lactobacillus curvatus, Lactobacillus fermentum; and the acidophilegroup comprising L. johnsonii; (see Fujisawa et al., Int. J. Syst.Bact., 42, 487-491, 1992); the pediococci (Pediococcus pentosaceus,Pediococcus acidilactici, and Pediococcus halophilus); the enterococci;the staphilococci (Staphylococcus xylosus and Staphylococcus carnosus);the micrococci (Micrococcus varians); yeast of the genus Debaromyces,Candida, Pichia, Torulopsis and Saccharomyces; and mold of the genusAspergillus, Rhizopus, Mucor and Penicillium.

The lactic bacteria according to the invention that are low acidifyingand capable of adhering directly to the pellicle of the teeth that areused to prepare compositions for the prophylaxis or the treatment ofdental caries, dental plaque, and periodontal infection displacepathogenic bacteria from the teeth or prevent the attachment of thepathogenic bacteria. The lactic bacteria according to the invention are“low acidifying,” which means that they are less acidifying thanpathogenic strains. Accordingly, they contribute to a pH in the oralcavity of about 5.5 to 7. Preferably, they are from dairy origin.

The lactic bacteria according to the invention adhere to the pellicle ofthe teeth via specific or unspecific interactions and/or adhesionfactors. The specific adhesion factors are proteins or polysaccharides.

At least one lactic bacteria is selected from the group consisting ofStreptococcus thermophilus, Lactococcus lactis subsp. lactis, andLactococcus lactis subsp. Lactis biovar diacetylactis and particularlyfrom the group consisting of the strains CNCM 1-1984, CNCM 1-1985, CNCM1-1986, CNCM 1-1987, and LMG P-18997. These strains have been selectedamong lactic bacteria strains for their capacity to adhere to thepellicle of the teeth and their optimal growth temperature of about 37°C., which is the temperature in the oral cavity. Moreover they arecapable of fermenting glucose and sucrose and do not synthesize glucans,which are factors leading to the pathogenicity of the cariogenicstrains.

In one embodiment of the invention the lactic bacteria are geneticallymodifying so that they adhere to the pellicle of the teeth via adhesionfactors. For lactic bacteria that already adhere to the pellicle of theteeth, this modification makes the strains more adherent to the surfaceof the teeth. In the same way, any non-adherent lactic acid bacteria(not Lactobacilli) can be genetically modified so that it adheres to thepellicle of the teeth. This modification of the lactic bacteria can beachieved, for example, by insertion of the genes X17390, X14490 orX53657 (GenBank accession numbers). These gene are responsible in S.mutans for the expression of the Antigen I/II that mediates adhesion tosalivary glycoproteins.

According to the invention, it is also possible to genetically modifylactic bacteria so that they are low acidifying. For lactic bacteriathat is already low acidifying this modification increases the effect byfurther decreasing lactic acid production. This modification can beachieved in many ways. Preferably, the modification is achievedaccording to one the protocols described in the following documents:Boumerdassi et al., Appl. Environ. Microbiol., 63, 2293-2299, 1997;Plattecuw et al., Appl. Environ. Microbiol, 61, 3967-3971, 1995; Ito etal., Biosci. Biotechnol. Biochem., 58, 1569-1573, 1994.

According to the invention, at least one lactic bacteria, geneticallymodified or not, is used in an “effective amount” for the preparation ofcompositions intended for the prophylaxis or the treatment of dentalcaries, dental plaque, and periodontal infection in humans or animalssuch as pets. This quantity is preferably between 10⁴ to 10⁹ cfu/g.

It is also possible to use the at least one lactic bacteria, incombination with milk derivatives, such as milk, fermented milk, or milkderivatives selected from any forms of caseino-glycomacropeptide,micellar casein, fluorinated micellar casein, renneted milk, orbacteriocin, for example.

Biochemical Characterization of the Selected Strains

Fermentation patterns: 49 simple sugars were tested with the api 50 CHbioMerieux strip test (bioMorieux SA, 69280 Marcy-l'Etoile, France). Theresults are given in the Table 1.

Acidification curves: Acidification curves were determined at 37° C.under the following conditions:

S. sobrinus OMZ 176: FUM sucrose 1% and FUM glucose 1%

S. thermophilus CNCM 1-1985: Belliker sucrose 1% and Belliker glucose 1%Inoculation was always 5%. The pH was recorded every 20 min.

S. thermophilus CNCM 1-1985, from sucrose fermentation, lowers the pH to4.5, while S. sobrinus OMZ 176 lowers the pH to 4. TABLE I Sugarfermentation of L. lactis CNCM I-1987, L. lactis CNCM I-1986, S.thermophilus CNCM I-1984, S. thermophilus CNCM I-1985 and, S.thermophilus LMG P-18997. L. lactis L. lactis S. th. S. th. S. th. SugarCNCM I-1987 CNCM I-1986 CNCM I-1984 CNCM I-1985 LMG P-18997 Adonitol +++Aesculin ++ ++++ Amygdalin ++++ D-Arabinose L-Arabinose D-ArabitolL-Arabitol +++ Arbutin +++ +++ Cellobiose +++ +++ Dulcitol ErythritolD-Fructose + ++++ D-Fucose L-Fucose Galactose ++ ++++ β-Gentiobiose +++Gluconate 2-keto-Gluconate 5-keto-Gluconate GlcNAc + ++++ D-Glucose +++++ + ++ ++ Glycerol Glycogen Inositol Inulin Lactose + ++++ +++ ++++++++ D-Lyxose Maltose ++ Mannitol +++ ++ D-Mannose + ++++ MelezitoseMelibiose α-Methyl-D-glucoside α-Methyl-D-mannoside D-Raffinose RhamnoseRibose ++ ++ Salicin +++ +++ Sorbitol L-Sorbose Starch Sucrose +++ +++++++ D-Tagatose Trehalose ++ D-Turanose ++ Xylitol +++ D-Xylose L-Xyloseβ-methil-xyloside+, ++, +++, ++++ show if the fermentation begins after 3, 6, 24, or 48hours, respectively.

The invention is also directed to compositions for the health of themouth that comprise a lactic bacteria that is not part of the residentmicroflora of the mouth, that is low acidifying, and that is capable ofadhering directly to the pellicle of the teeth. The compositions areparticularly intended for the prophylaxis or the treatment of dentalcaries, dental plaque, and periodontal infection. The lactic bacteriastrain may be selected from the group consisting of Streptococcusthermophilus, Lactococcus lactis subsp. lactis, and Lactococcus lactissubsp. lactis biovar diacetylactis and preferably from the groupconsisting of the strains CNCM I-1984, CNCM I-1985, LMG P-18997, CNCMI-1986, and CNCM I-1987. In these compositions the lactic bacteriastrains may be genetically modified as described above.

The lactic bacteria strains may be included in a food, pet food,cosmetic, or pharmaceutical composition, for example. Accordingly, thecompositions are preferably a toothpaste, mouth rinse, gum, spray,beverage, candy, infant formula, ice cream, frozen dessert, sweet saladdressing, milk preparation, cheese, quark, yogurt, acidified milk,coffee cream, or whipped cream, for example.

In the compositions of the invention, the lactic bacteria strains may beincluded alone or in combination with milk derivatives, for example, inorder to obtain synergistic preparations. Accordingly, thesecompositions for the health of the mouth comprise:

a lactic bacteria that is not part of the resident microflora of themouth, which is capable of adhering directly to the pellicle of theteeth;

any forms of lactic glycopeptides, renneted milk, or bacteriocin.

The lactic glycopeptides are preferably caseino-glycomacropeptides(CGMP), fluorinated or non-fluorinated micellar casein (which can beobtained as described in EP 0 604 802 and EP 0 748 591), or rennetedmilk. The caseino-glycomacropeptides are preferably added in a minimumamount of about 0.1%. It has also been shown that thecaseino-glycomacropeptides do not prevent the lactic bacteria fromadhering to the teeth pellicle (FIGS. 2 and 3).

Synergistic compositions may also be prepared by adding at least onebacteriocin which is active against Gram-positive oral bacteria. In thisembodiment the oral hygiene compositions may comprise 0.00001 to 50%,and preferably from 0.00001 to 15% of purified bacteriocin, by weight ofthe composition. The bacteriocin is preferably variacin (EP 0759469).

To protect the composition from degradation, an oil-soluble antioxidantmay also be included. Suitable antioxidants include the “tocopherols,”butyl-hydroxyanisole (BHA), butyl-hydroxytoluene (BHT), and ascorbylpalmitate. The oil soluble antioxidant is present in amounts of from0.005% to 0.5%, preferably 0.005% to 0.01% by weight of the composition.

Suitable abrasives for use in dentifrice compositions of the presentinvention include calcium carbonate, calcium aluminosilicate, aluminahydrates, alumina, zinc orthophosphate, plastic particles, and silica,of which silica is the preferred abrasive.

Compositions according to the invention will have a pH which is orallyacceptable and within a range such that the activity of the lacticbacteria is not compromised. The pH may be in the range of 3.0 to 9.5,preferably in the range 3.5 to 6.5.

The compositions of the invention may be prepared by conventionalprocesses that comprise admixing the ingredients together in theappropriate relative amounts and finally, if necessary, adjusting the pHto the desired value.

The invention is further directed to a method for screening lacticbacteria capable of adhering to tooth. This method comprises the stepsof:

-   (1) preparing monoclonal antibodies that recognize specific surface    proteins of a lactic bacteria strain capable of adhering to the    teeth, and-   (2) screening any lactic bacteria strain by using the monoclonal    antibody of strain capable of adhering to the teeth.

The monoclonal antibodies are used as a tool to detect the said lacticbacteria strain among other strains growing nearby.

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention, in addition to those described herein, will become apparentto those skilled in the art from the foregoing description andaccompanying figures. Such modifications are intended to fall within thescope of the claims. Various publications are cited herein, thedisclosures of which are incorporated by reference in their entiretiesto the extent necessary for understanding the present invention. DNAmanipulation, cloning and transformation of bacteria cells are, exceptwhere otherwise stated, carried out according to the textbook ofSambrook et al. (Sambrook et al., Molecular Cloning, A LaboratoryManual, Cold Spring Harbor Laboratory Press, U.S.A., 1989).

EXAMPLES

The examples are preceded by a brief description of the plasmids,strains, and the various media used, as well as the method for producinga monoclonal antibody.

The strains S. thermophilus S118 (NCC 1529), S123 (NCC 1561), L. lactissubsp. Lactis 29 (NCC 2211), L. lactis subsp. lactis biovardioacetylactis 69 (NCC 2225) were deposited under the Budapest Treaty atthe Collection Nationale de Culture de Microorganismes (CNCM 1-1984,CNCM 1-1985, CNCM 1-1986 and CNCM 1-1987, respectively), 25 rue dudocteur Roux, 75724 Paris, France, on Mar. 3, 1998. The strain S.thermophilus BFI 1116 (CNBL 1177) was deposited under the BudapestTreaty at the Belgian Coordinated Collections of Microorganisms LMGP-18997, K. L. Ledeganckstraat 35, B-9000 Gent, Belgium, on Jul. 5,1999. All restrictions as to the availability of these deposits will bewithdrawn upon first publication of this application or anotherapplication which claims benefit of priority to this application.

Example 1 Strains and Culture Conditions

More than 100 strains (belonging to the Nestle culture collection) werescreened for their ability to attach to saliva-coated hydroxyapatitebeads, and in particular the following 23 strains: S. thermophilus Y54(NCC 2284), S. thermophilus 5fi6 (NCC 1971), S. thermophilus Sfi13 (NCC2008), S. thermophilus Sfi21 (NCC 2038), S. thermophilus Sfi39 (NCC2130), S. thermophilus Sfi42 (NCC 2145), S. thermophilus Sfi47 (NCC2172), S. thermophilus S118 (NCC 1529), S. thermophilus S119 (NCC 1536),S. thermophilus S122 (NCC 1554), S. thermophilus S123 (NCC 1561), S.thermophilus S126 (NCC 1587), L. lactis subsp. cremoris 15 (NCC 92), L.lactis subsp. cremoris 25 (NCC 1932), L. lactis subsp. cremoris 136 (NCC2419), L. lactis subsp. diacetylactis 8 (NCC 1970), L. lactis subsp.diacetylactis 28 (NCC 2057), L. lactis subsp. diacetylactis 69 (NCC2225), L. lactis subsp. diacetylactis 80 (NCC 2272), L. lactis subsp.lactis 29 (NCC 2211), L. lactis subsp. lactis 50 (NCC 2224), L. lactissubsp. lactis 54 (NCC 2228), S. macedonicus 216 (NCC 2484).

The 5 oral strains, S. sobrinus OMZ 176, S. oralis OMZ 607, A.naeslundii OMZ 745, V. dispar OMZ 493 and F. nucleatum OMZ 596 wereobtained from the Institute für Orale Mikrobiologie und AligemeineImmunologie, University of Zürich and were cultured in FUM medium inanaerobiosis (GasPackSystem, BBL) at 37° C.

All the strains were stored in glycerol at −20° C. and pre-cultured for14 hours prior to use at their specific optimal temperature; S. sobrinusOMZ 176 grew in FUM medium lactococci and streptococci in M17 (Difco)except S. thermophilus NCC1529, S119, S122, NCC1561 and S126 that grewin Belliker (prepared by dissolution of 20 g tryptone, 5 g yeastextract, 2.5 g gelatine, 5 g dextrose, 5 g sucrose, 5 g lactose, 4 gNaCl, 0.5 g Ascorbic acid, and 10 g beef extract in 1 L of water).

For plate counting, S. sobrinus OMZ 176 was cultured in Mitis-Salivariusagar (Difco), S. thermophilus NCC1529, S119, S122, NCC1561, BFI 1116,and S126 in Belliker agar (prepared by adding to liquid Belliker 15 g ofBacto agar, Difco), and the remaining lactic bacteria strains in M17agar (Oxoid).

Example 2 Production of Monoclonal Antibody

A monoclonal antibody would be used as a tool to detect L. lactis subsp.lactis NCC2211 among 5 oral strains growing together on S-HA discs andforming a biofilm that simulates dental plaque. Therefore the monoclonalantibody was tested against these strains to verify there was nocross-reaction. To this end, the monoclonal antibody is produced asdescribed by Granato et al. “A mouse monoclonal IgE antibody anti-bovinemilk lactoglobulin allows studies of allergy in the gastrointestinaltract., Clin. Exp. Immunol., 63, 703-710, 1986.

Example 3 Selection of Adherent Lactic Bacteria

Attachment to Saliva-Coated Hydroxyapatite Beads (S-HA)

To select among the lactic bacteria dairy strains those strains that areable to attach to saliva-coated hydroxyapatite beads (S-HA), theprocedure previously described by Neeser et al. (1994) was used withslight modification in that the bead washings were done with 150 μlvolumes and Hyamine hydroxide was substituted with Benzethoniumhydroxide (Sigma).

Briefly, all the strains were grown to the end of the log phase in FUMexcept S. thermophilus NCC1529, S119, S122, NCC1561, and S126 that werecultured in Belliker. S. sobrinus OMZ 176, L. lactis subsp. lactisNCC2211, 50 and 54, S. thermophilus NCC1529, S119, S122, NCC1561, andS126 grew at 37° C., the remaining lactococci at 30° C., and theremaining streptococci at 42° C.

5 mg of hydroxyapatite beads (BDH Chemicals Ltd, Poole, England) werecovered with 70 μl clarified saliva obtained from volunteers in the laband prepared as previously explained (Neeser et al, 1994). Saliva coatedbeads were kept overnight at 4° C., then washed (first with distilledwater and after with HEPES buffer) and finally inoculated with 100 μl ofmetabolically labeled bacterial suspension (bacteria had been grown inmedium supplemented with 10 μCi/ml ¹⁴C acetic acid). Adhesion took placeduring 45 min at 37° C., then unbound bacteria were washed away and theattached cells directly counted in a LKB scintillation counter (type1219 Rackbeta).

Adhesion percentages are expressed as radioactivity bound to the beadsrelative to the total radioactivity added to each well. All measurementswere done in triplicate. Table 2 reports the percentages of adhesion tosaliva-coated hydroxyapatite beads obtained for several screened strainsand for S. sobrinus OMZ 176 (the reference strain). TABLE 2 Percentagesof Adhesion to Saliva-coated Hydroxyapute Beads for Several ScreenedStrains. STRAIN % ADHESION (±SD) S. sobrinus OMZ 176 2.23 ± 0.49 S.thermophilus Sfi42 (NCC 2145) 0.08 ± 0.02 S. thermophilus Sfi47 (NCC2172) 0.14 ± 0.04 S. thermophilus NCC1529 2.89 ± 0.60 S. thermophilusS119 (NCC 1536) 0.15 ± 0.04 S. thermophilus S122 (NCC 1554) 0.93 ± 0.17S. thermophilus NCC1561 2.19 ± 0.50 S. thermophilus S126 (NCC 1587) 1.19± 0.56 L. lactis subsp. diacetylactis 28 (NCC 2057) 1.59 ± 0.17 L lactissubsp. diacetylactis NCC2225 1.96 ± 0.40 L. lactis subsp. diacetylactis80 (NCC 2272) 1.20 ± 0.35 L lactis subsp. lactis NCC2211 2.85 ± 0.85

Four strains, S. thermophilus NCC 1529 (CNCM 1-1984), S. thermophilusNCC1561 (CNCM 1-1985), L. lactis subsp. lactis NCC2211 (CNCM 1-1986)(hereinafter L. lactis NCC2211) and L. lactis subsp. diacetylactisNCC2225 (CNCM 1-1987) showed adhesion values close to S. sobrinus OMZ176.

L. lactis NCC2211 and S. thermophilus NCC1561 were chosen as the morepromising candidates since they grow very well at 37° C., which is thetemperature in the mouth, while L. diacetylactis NCC2225 has an optimalgrowth temperature of 30° C. In particular, L. lactis NCC2211 cannotgrow on sucrose, but it can ferment a wide range of sugars, moreoverother oral strain can provide glucose via their invertase.

Adhesion Saturation Curves

Curves of bound CFU versus CFU inoculated into the well were determinedto verify if bead saturation could be obtained. The 50% saturation wasobtained directly from the bending point of the curves. The adhesionsaturation curves for S. sobrinus OMZ 176, L. lactis NCC2211, and S.thermophilus NCC 1561 were determined. They are shown in FIG. 1.

For each of the three strains the CFU number inoculated in the well toget 50% bead saturation and the corresponding number of bound CFU weredirectly deduced from the bending point of the curves and are given inthe table 3. TABLE 3 Number of CPU Inoculated Per Well to get 50% BeadSaturation. cfu/well Bound cfu % adhesion S. sobrinus OMZ 176 4.00E+074.00E+06 10%  L. lactis NCC2211 1.00E+07 9.00E−f-05 9% S. thermophilusNCC156I 3.00E+07 2.00E+06 7%

Example 4 Effect of Caseino-Glycomacropeptides

The influence of CGMP on the adhesion of L. lactis NCC2211 and S.thermophilus NCC 1561 was studied to verify the possibility of usingCGMP to foster the predominance of one of these two strains overpathogenic strains, namely S. Sobrinus OMZ 176. Caseino-glycopeptide(CGMP) and its desialylated derivative (As-CGMP) were obtained fromNestec S. A., Lausanne (for their preparation see Neeser et al., 1994).

The dose-response effect was studied on the adhesion to S-HA beads byinoculating, in the well, 100 μl of bacterial suspension (CFU/mlcorresponding to the 50% bead saturation previously calculated) whichcontained CGMP or AsCGMP in different concentrations and then performingthe adhesion assay in the usual manner. Concentrations in the range 0.05to 3 mg/ml were tested. No previous incubation of the bacteria inpresence of CGMP or As-CGMP was done.

FIG. 2 provides the curves obtained for the three strains by plottingthe number of bound cells versus increasing amounts of CGMP, the numberof inoculated cells corresponds to 50% bead saturation formerlycalculated for each strain. The strong inhibition observed in the caseof S. sobrinus OMZ 176 confirms the previous results obtained by Neeseret al. (1994) and Schupbach et al. (J. Dent. Res., 75, 1779-1788, 1996).

FIG. 2 shows that 0.25 mg/ml produced 50% inhibition of the adhesion ofS. sobrinus OMZ 176, while more than 2 mg/ml were necessary to have thesame effect with S. thermophilus NCC1561. CGMP slightly enhances theadhesion of L. lactis NCC2211.

As in the case of CGMP, the desyalilated derivative inhibits theadhesion of S. sobrinus OMZ 176; only 0.05 mg/ml are needed to produce50% decrease in the adhesion percentage. As-CGMP does not influence L.lactis NCC2211 adhesion, while it slightly fosters the adhesion of S.thermophilus NCC1561 (FIG. 3).

Example 5 Toothpaste

Toothpaste is prepared by adding 10⁵ cfu/ml of at least one of thelactic bacteria strain CNCM 1-1984, CNCM 1-1985, CNCM 1-1986, CNCM1-1987 or LMG P-18997 in a lyophilized form, to a mixture containing:Cetyl pyridinum chloride 1.65% Sorbitol (70% soln) 33.0% Glycerin 25.0%Sodium carboxymethyl cellulose  2.0% Sodium fluoride 0.25% Silica (RP93) 26.3% Thickening Silica (Sident 22)  8.1% Sodium saccharine  0.5%Poloxamer (Pluronic F 108)  3.2%

The toothpaste is intended for the prophylaxis or the treatment ofdental caries, dental plaque, and periodontal infection.

Example 6 Ice Cream

A cream comprising 10.8% lactic fats, 13.5% milk solids (non fat), 0.3%Emulstab® SE30 and 0.3% Emulstab® foam (Grindsted, DK) is prepared andthen pasteurized at 105° C. for 20s, homogenized at 75° C. and 300 bar,cooled to 38° C., and inoculated with pre-cultures in MRS medium, takenin the exponential growth phase, at a rate of 10⁷ to 10⁸ cfu/ml of atleast one of the lactic bacteria strain of CNCM 1-1984, CNCM 1-1985,CNCM 1-1986, CNCM 1-1987 or LMG P-18997. The cream is then fermented for10 hours at 38° C. up to a pH of about 4.5. At the end of thefermentation, sucrose and glucose syrup is added thereto. Thecomposition of the cream is presented in table 4 below. The mixture isthen beaten, cooled to 4° C., stored at 4° C., and chilled to a degreeof expansion of 95° C. by volume. TABLE 4 Ice Cream Composition Non-fatSolids Composition Fats solids Sucrose content Ingredients (kg) (%) (%)(%) (%) Cream (35%) 30.83 10.79 1.54 12.33 Powdered 12.45 11.95 11.95skimmed milk Emulstab ® 5E30 0.41 0.37 Emulstab ® foam 0.41 0.36 Water55.91 Total: cream base 100.00 10.79 13.49 — 25.01 Cream base 74.14 8.0010.00 — 18.54 Sucrose 22.06 15.00 15.00 Glucose syrup 3.80 3.00Fermented 100.00 8.00 10.00 15.00 36.54 Ice cream

Example 7 Yogurt

5 L MRS culture medium were sterilized for 15 min at 121° C. and theninoculated with 5% by volume of an active culture of at least one of theS. Thermophilus strains CNCM 1-1984, CNCM 1-1985, or LMG P-18997containing approximately 10⁹ cfU/ml. After incubation for 8 h at 41° C.,a starter containing 4.5×10⁸ cfu/ml was obtained.

5 L of reconstituted skimmed milk having a dry matter content of 10%, towhich 0.1% yeast extract had been added, was sterilized for 15 min at121° C. and inoculated with 2% of an active culture of commercialthickening Streptococcus thermophilus containing approximately 10⁹cells/ml. After incubation for 4 h at 41° C., a starter containing4.5×10⁸ cells/ml was obtained.

One batch of whole milk containing 3.7% fats strengthened with 2.5%skimmed milk powder and then pasteurized for 30 min at 90° C. was theninoculated with 2% by volume of the starter of at least one of the CNCM1-1984, CNCM 1-1985 or LMG P-18997 strains and 3% by volume of thestarter of thickening Streptococcus thermophilus. The inoculated milk isstirred, poured into pots, and incubated for 4 h at 41° C. The resultingyogurt obtained has a good firm and smooth texture and is intended forthe health of the mouth.

Example 8 Chewing Gum

A chewing gum for preventing or treating dental caries, dental plaque,or periodontal infection can be prepared adding an active culture of atleast one of the S. Thermophilus strains CNCM 1-1984, CNCM 1-1985, orLMG P-18997 so that it contains approximately 10⁴ to 10⁹ cfu/g, to thefollowing typical ingredients: Xylitol 67.5%   Gum base 20%  Calciumcarbonate 5% Glycerin 3% PluronicFl27 2% Cellulose gum 1% Ballastcompounds 0.5%   Flavor 1%

Example 9 Pet Food Composition

A pet food for mouth health is obtained by preparing a feed mixture madeup of corn, corn gluten, chicken and fish meal, salts, vitamins, andminerals. The feed mixture is fed into a pre-conditioner and moistened.The moistened feed leaving the pre-conditioner is then fed into anextruder-cooker and gelatinised. The gelatinised matrix leaving theextruder is forced through a die and extruded. The extrudate is cut intopieces suitable for feeding to dogs, dried at about 110° C. for about 20minutes, and cooled to form pellets which have a water activity of about0.6. The pellets are sprayed with 3 coating mixtures. Each coatingmixture contains an active culture of at least one of the S.Themmophilus strains CNCM 1-1984, CNCM 1-1985, or LMG P-18997 but onecoating mixture uses hydrogenated soy fat as a coating substrate, onecoating mixture uses water as a coating substrate, and one coatingmixture uses protein digest as a coating substrate. The pellets containapproximately 10⁴ to 10⁹ cfu/g of the strains.

1. A method of treating or preventing dental caries, dental plaque, andperiodontal infection in humans or animals comprising administering tothe oral cavity of a human or animal one or more lactic bacteria thatare not part of the resident microflora of the human mouth, that are lowacidifying in that when administered they provide a pH in the oralcavity of 5.5 to 7, and that are capable of adhering directly to thepellicle of the teeth to displace from the teeth or prevent attachmentto the teeth of cariogenic strains of bacteria that are residentmicroflora of the mouth.
 2. The method of claim 1, wherein the lacticbacteria are originally derived from a diary.
 3. The method of claim 1,wherein the lactic bacteria comprise one or more of Streptococcusthermophilus,llactococcus lactis subsp. lactis, or Lactococcus lactissubsp. lactis biovar diacetylactis.
 4. The method of clam 1, wherein thelactic bacteria have optimal growth at a temperature of 30° C. to 42° C.5. The method of claim 1, wherein the lactic bacteria have beengenetically modified to have improved adherence to the pellicle of theteeth by insertion of the X17390 gene, the X14490 gene, or the X53657gene.
 6. The method of claim 1, further comprising administering thelactic bacteria in combination with one or more of milk, fermented milk,milk derivatives, or bacteriocin.
 7. The method of claim 6, wherein themilk derivative comprises one or more of a caseino-glycomacropeptide,micellar casein, fluorinated micellar casein, or renneted milk.
 8. Themethod of claim 1, wherein the lactic bacteria that are used fortreating or preventing dental caries are administered by way of acomposition that contains the lactic bacteria in an amount of 10⁴ to 10⁹cfu/g in order to provide the pH of at least 5.5 when the composition isadministered to the oral cavity of a human or animal.
 9. The method ofclaim 8, wherein the composition further contains one or more of milk,fermented milk, or a milk derivative.
 10. The method of claim 9, whichfurther comprises a bacteriocin in an amount of 0.00001 to 50 percent byweight of the composition.
 11. The method of claim 10, wherein thecomposition includes a milk derivative comprising one or more of acaseino-glycomacropeptide, micellar casein, fluorinated micellar casein,or renneted milk in an amount of at least 0.1 percent by weight of thecomposition.
 12. The method of claim 8, wherein the composition furthercomprises one or more of an oil soluble antioxidant or an abrasive. 13.The method of claim 8, wherein the composition is in the form of atoothpaste, mouth rinse, gum, spray, beverage, candy, infant formula,ice cream, frozen dessert, sweet salad dressing, milk preparation,cheese, quark, yogurt, acidified milk, coffee cream or whipped cream.